Polysomy 21 (extra copies of chr.21) is the most common aneuploidy in B-cell acute lymphoblastic leukemia (B-ALL). Constitutional trisomy 21 (Down Syndrome, DS) is associated with a 20-fold increased risk of B-ALL, strongly suggesting a causal link. Polysomy 21 is also the most common somatically-acquired aneuploidy in B-ALL, including the poor-risk subsets with BCR-ABL and CRLF2 rearrangements. Yet, the mechanisms underlying this association are unknown. We previously identified an in vitro transformed phenotype among precursor B-cells from Ts1Rhr mice, which are trisomic for only the 33 genes within the Down Syndrome Critical Region (DSCR). Specifically, B-lineage cells from Ts1Rhr mice have increased colony formation and indefinite serial replating potential in methycellulose culture. We now demonstrate that both Ts65Dn mice, which harbor a larger triplication of genes syntenic with human chr.21, and Ts1Rhr mice have a defect in B- lineage ontogeny at the Hardy A to Hardy B transition. In addition, DSCR trisomy promotes in vivo B-cell leukemogenesis in concert with BCR-ABL. Transcriptome sequencing of Ts1Rhr and wild-type B-cells identified a signature from DSCR trisomy that is highly associated with targets of the polycomb repressor complex 2 (PRC2) and its target, trimethylated lysine 27 on histone H3 (H3K27me3). Both gain- and loss-of- function mutations in PRC2 components are common in lymphoid malignancies but were not recurrently identified in B-ALL. An shRNA screen to identify DSCR loci that suppress serial replating potential in Ts1Rhr B-cells implicated HMGN1 (high mobility group nucleosome binding domain 1), a nucleosome remodeling protein that increases chromatin accessibility, enriches at active promoters and may suppress H3K27me3. We will build on these discoveries to define the mechanisms that promote B-ALL in cells with polysomy 21 and identify new therapeutic targets in this disease. In Aim 1, we will utilize agnostic approaches to define the transcriptional and epigenetic alterations underlying in vitro and in vivo B-lineage phenotypes in both primary B-cells and B-ALL. In Aim 2, we will specifically address the hypothesis that DSCR trisomy promotes B-ALL through alterations in H3K27me3. Chr.21 is the second most commonly gained chromosome in acute myelogenous leukemia, suggesting a broader significance for these studies beyond B-ALL. Finally, this project utilizes innovative approaches to define and therapeutically target the biologic consequences of recurrent copy number alterations, a nearly ubiquitous finding in cancer.